Classification

Higher order taxa

Species

Description and significance

Lactococcus lactis is a spherical-shaped, Gram-positive bacterium used widely for industrial production of fermented dairy products such as milk, cheese, and yogurt. These are important food supplies for many people, so extensive research has been done on the microorganism’s metabolic pathway to increase its efficiency for dairy production. Due to its important applications, simple metabolism and limited biosynthetic capabilities, its genome has been sequenced to help researchers understand genes that are responsible for its fermentation pathway. Furthermore, scientists study DNA recombination to improve its survival and resistance to antibiotics and to manipulate its metabolic pathways for better use in industrial production. (10)

Aside from its high use in industrial application, Lactococcus lactis can also be found in the wild on plants and within the digestive tract of cows. It is believed that in nature, L. lactis stays dormant on plant surfaces awaiting to be ingested along with the plant into animal gastrointestinal tract, where it becomes active and multiplies intensively (1). Not only it is important in dairy production, it also has potential of use as oral vaccine, foreign protein production and metabolite through genetic engineering to manipulate L. lactis in the researchers’ favor (2).

Lactococcus lactis has two subspecies with few phenotype and genotype differences, Lactococcus lactis subsp. lactis and subsp. cremoris, where subsp. lactis is preferred for making soft cheese while subsp. cremoris is for hard cheese (1). These organisms were originally classified under the genus Streptococcus, but in 1985, it was assigned to the current genus (3). Recently, one method of distinguishing between the two subspecies was announced. Observation of glutamate decarboxylase (GAD) can be found in the subspecies lactis, but not in subspecies cremoris. “GAD catalyzes the irreversible decarboxylation of glutamate to gamma-aminobutyric acid” with the glutamate-GABA antiporter (GABA) (4). The gene that encodes GAD in L. lactis subsp. cremoris is inactivated by a frameshift mutation resulting in a nonfunctioning protein.

Genome structure

The genome of L. lactis is a circular chromosome with 2,365,589 base pair, where 86% of the genome code for protein, 1.4% for RNA, and 12.6% for noncoding region. 64.2% of the genes code for known functional proteins and 20.1% of the genes for known protein with unknown function. The remaining 15.7% of the genes are unidentified proteins that may be unique to the Lactococcus. The DNA replication initiation genes in L. lactis includes dnaB, dnaD, and dnaI; in addition, there are two DNA polymerase III encoded by the polC and dnaE genome. Furthermore, there are around 30 genes for transcription and only 3 sigma factor involved in reading the DNA. Translation is more intricate including 119 genes while 27 genes are utilized for protein fixation after translation. (1)

Cell structure and metabolism

The metabolic pathway of L. lactis can function through aerobic and anaerobic reactions. It consists of 621 reactions and 509 metabolites and requires minimally glucose, arginine, methionine, glutamate and valine for growth. The main metabolism of L. lactis is through the anaerobic pathway, fermentation, which produces lactic acid from the available carbohydrates and is used for industrial food production. The carbon sources that L. lactis draws from include fructose, galactose, glucosamine, glucose, lactose, maltose, mannitol, mannose, ribose, sucrose and trehalose. However, the growth rate of the cell with the intake of each carbon source is different. Growth rate on glucose, mannose, galactose, sucrose, lactose and glucosamine are the same, while fructose and mannitol growth rates are lower. (5)

Under anaerobic reaction, glycolysis breaks down extracellular carbohydrates to pyruvate, then converts pyruvate to lactic acid with the main enzyme ldh, lactate dehydrogenase (6). Lactate will guide protons out with membrane protein creating the membrane potential necessary for energy production (1). NADH, the cofactor for the lactate dehydrogenase is regenerated into NAD+ to be reused for glycolysis. This mechanism allows L. lactis to survive without the genes for citric cycle and gluconeogenesis.

Besides from the anaerobic pathway, L. lactis has an aerobic system to assist in its development. Normally, the uptake of oxygen would affect the fermentation process or even interferes with oxygen reactive substances. However under low oxygen consumption, aerobic pathway is limited due to the low recycle rate of NAD from NADH oxidase. These damages can be fixed when cells are grown with oxygen and heme source. Cells increase their growth, resistance to oxidation, and improve survival at low temperature. Furthermore, it has recently been discovered that a functional electron transfer chain in L lactis supports the membrane potential. Along with heme source, the presence of membrane bound NADH dehydrogenase oxidizes NADH and increases the cell growth and production of proteins and vitamins. (7)

Pathology

Lactococcus lactis is nonpathogenic bacteria.

Application to Biotechnology

Lactococcus lactis is researched thoroughly and put into many applications. It has several fermentative pathways, but the most important purpose is its property to manufacture dairy product such as cheese and milk. Lactococcus lactis specializes in lactate dehydrogenase excreting lactic acid, which is used to preserve food and extend food shelf life. Dairy industries continue to improve the activities and effectiveness of L. lactis by manipulating its environment and cell behavior.

Another study utilizes the simple and harmless bacteria as mucosal vaccine. In developing countries where vaccines are limited and not affordable, diseases can spread easily. Researchers attempt to prove that a mucosal vaccine against Streptococcus pneumoniae using Lactococcus lactis is more effective than vaccination with purified live antigen. L. lactis is treated to recombine vaccine strains, so the cell can express the vaccine protein PspATIGR4. The result shows that L. lactis has more potential and safety in developing vaccine in human and should be considered to be used against more pathogens. (6)

Current Research

1. Recombining genes has been a difficult issue, so in recent research, scientists demonstrate the parameters required for successful transformation of L. lactis IL1403 with electroporation. The resistors used in electroporation and the pulse decay time are important factors. The concentration of 2-3% glycine in the media also will provide the optimal growth for the transformation efficiency. This research will help the dairy industry to manipulate the genome to have better control over the organism’s activities. (8)

2. Most current research deals with improving the industrial use of L. lactis. A new method optimizing lactic acid fermentation has been studied recently that deals with immobilizing the cells to provide advantages. The cells are first entrapped within beads of alginate or microcapsules of alginate membrane. The advantage from this method includes the ability to reuse the immobilized cells shortening the processing time, elimination of process to remove the bacteria from the final product, high density of cell to increase activity and production, and reduce contamination. As a result, optimal lactic acid production can be achieved with the immobilized group of L. lactis and continuous operation with controlled pH. However, the major downsides to this method consist of the cost of creating batch of immobilized cells and the cost of fermentation medium. (9)

3. Another industrial research on L. lactis deals with the production of l-alanine, which is used as sweetener in dairy products. Many microorganism produce l-alanine, but the maximum conversion rate from carbohydrates remain only between 50–60%. The alaD gene coding for alanine deydrogenase, an enzyme that converts pyruvate into alanine, was inserted into L. lactis chromosome. To ensure for maximal production, lactate dehydrogenase gene was knocked out to eliminate any competition for pyruvate substrate and cofactors. When this cell is grown under uncontrolled pH, it produces 50% alanine, 35% acetoin, and other substances. In addition, controlling the pH to 7.5, the mutant cell improves the production to 75% alanine. The alanine production with change of pH increases drastically over the maximal conversion of other microorganism. The increase in performance of L. lactis reveals another industrial advantage for producing amino acid and genetic engineering. (6)